Antiretroviral therapy (ART) is highly effective at suppressing HIV-1 replication but does not eliminate infected cells. Host immune responses may influence viral reservoir cell evolution, but such selection effects may only become detectable after prolonged durations of ART. We conducted a high-resolution cross-sectional and longitudinal analysis of the proviral reservoir landscape in long-term ART-treated (LT-ART) patients.
Chromosomal integration sites of intact and defective proviruses were analyzed in people living with HIV (n=8) who had remained on suppressive ART for approximately 20 years, using full-length individual proviral sequencing (FLIP-Seq) and matched integration site and proviral sequencing (MIP-Seq). Corresponding data from individuals with shorter durations of ART (median 9 years) (n=43) were used as a reference cohort.
In total, 612 proviral genomes (277 intact, 335 defectives) were obtained; 106 integration sites (64 unique) of intact proviruses and 128 integration sites (85 unique) of defective proviruses were identified. After approximately 20 years of treatment, LT-ART individuals did not show a significant difference in the frequency of total or intact HIV-1 proviruses compared to individuals with shorter durations of suppressive ART; however, the proportion of clonally-expanded intact proviruses was significantly higher (83% vs 42%, p < 0.0001). Intact proviruses from LT-ART patients showed a highly biased chromosomal integration site profile, with 38%, 23% and 23% of intact genomes being integrated in centromeric/satellite DNA, in non-genic DNA and in ZNF genes, respectively. This represented an apparent contrast to patients with shorter ART duration, in whom 0%, 18% and 13% of intact proviruses were detected in centromeric/satellite DNA, non-genic DNA and ZNF genes, respectively. No difference in chromosomal integration site locations were noted for defective proviruses between the two cohorts. Longitudinal evaluations in n=5 study participants demonstrated a progressive accumulation of intact proviruses in non-genic DNA in LT-ART relative to earlier stages of treatment (15% after 1-2 years ART vs 61% after 17-20 years ART, p=0.002).
Long-term ART is associated with progressive enrichment of intact proviruses integrated in repressive heterochromatin locations, likely as a result of immune-mediated selection mechanisms that preferentially eliminate proviruses in more transcriptionally-permissive chromosomal regions.